An Inducible Cell-Cell Fusion System with Integrated
Ability to Measure the Efficiency and Specificity of HIV-1
Alon Herschhorn1, Andres Finzi1, David M. Jones2, Joel R. Courter2, Akihiro Sugawara2, Amos B.
Smith III2, Joseph G. Sodroski1,3*
1Department of Immunology Cancer and AIDS, Dana-Farber Cancer Institute and Department of Microbiology and Immunobiology, Harvard Medical School, Boston,
Massachusetts, United States of America, 2Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 3Department of
Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America
HIV-1 envelope glycoproteins (Envs) mediate virus entry by fusing the viral and target cell membranes, a multi-step process
that represents an attractive target for inhibition. Entry inhibitors with broad-range activity against diverse isolates of HIV-1
may be extremely useful as lead compounds for the development of therapies or prophylactic microbicides. To facilitate the
identification of such inhibitors, we have constructed a cell-cell fusion system capable of simultaneously monitoring
inhibition efficiency and specificity. In this system, effector cells stably express a tetracycline-controlled transactivator (tTA)
that enables tightly inducible expression of both HIV-1 Env and the Renilla luciferase (R-Luc) reporter protein. Target cells
express the HIV-1 receptors, CD4 and CCR5, and carry the firefly luciferase (F-Luc) reporter gene under the control of a tTA-
responsive promoter. Thus, Env-mediated fusion of these two cell types allows the tTA to diffuse to the target cell and
activate the expression of the F-Luc protein. The efficiency with which an inhibitor blocks cell-cell fusion is measured by a
decrease in the F-Luc activity, while the specificity of the inhibitor is evaluated by its effect on the R-Luc activity. The system
exhibited a high dynamic range and high Z’-factor values. The assay was validated with a reference panel of inhibitors that
target different steps in HIV-1 entry, yielding inhibitory concentrations comparable to published virus inhibition data. Our
system is suitable for large-scale screening of chemical libraries and can also be used for detailed characterization of
inhibitory and cytotoxic properties of known entry inhibitors.
Citation: Herschhorn A, Finzi A, Jones DM, Courter JR, Sugawara A, et al. (2011) An Inducible Cell-Cell Fusion System with Integrated Ability to Measure the
Efficiency and Specificity of HIV-1 Entry Inhibitors. PLoS ONE 6(11): e26731. doi:10.1371/journal.pone.0026731
Editor: Mark Wainberg, McGill University AIDS Centre, Canada
Received August 16, 2011; Accepted October 3, 2011; Published November 1, 2011
Copyright: ? 2011 Herschhorn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: A.F. was supported by an American Foundation for AIDS Research Mathilde Krim Fellowship in Basic Biomedical Research # 107431-45-RFRL. A.S. was
supported by a Japan Society for the Promotion of Science (JSPS) research fellowship. The study was supported by the National Institutes of Health (grants
GM56550, AI24755, AI67854 and AI60354), the International AIDS Vaccine Initiative, and the late William F. McCarty-Cooper. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that
causes acquired immunodeficiency syndrome (AIDS) in humans.
HIV-1 establishes a persistent infection that, in the absence of
treatment, results in the severe depletion of CD4-expressing
lymphocytes and usually fatal immunodeficiency [1,2]. Antiretroviral
therapy for HIV-1 infection combines inhibitors against several
protease, gp41 and integrase, and also includes a ligand of the CCR5
co-receptor that blocks viral entry . The use of a combination of
drugs efficiently decreases virus loads and extends the lifespan of
HIV-1-infected individuals. However, despite the large and effective
arsenal available to fight HIV-1, resistant variants of HIV-1
eventually evolve during therapy; moreover, some antiretroviral
drugs exhibit long-term toxicity [3,4,5,6,7,8]. Thus, it is essential to
identify additional new inhibitors with low cytotoxicity and broad-
range activity against diverse HIV-1 strains for future success in
inhibitors may be also used to prevent HIV-1 transmission. This
strategy has been validated in the recent partial success of tenofovir, a
reverse transcriptase inhibitor, in preventing sexual transmission of
HIV-1 when it was administrated either orally or as a topical
The HIV-1 envelope glycoproteins (Envs) mediate virus entry
into cells, and represent attractive targets for intervention. Three
gp120 exterior Envs and three gp41 transmembrane Envs are
assembled into the trimeric envelope spike and anchored on the
HIV-1 virion surface by the gp41 membrane-spanning segments
[11,12,13]. The gp120 glycoprotein binds the CD4 receptor and
either the CCR5 or CXCR4 chemokine coreceptor [14,15,16,17].
Receptor binding moves the high-potential-energy Env complex
into lower-energy forms, culminating in the formation of a six-
helix bundle in gp41 that mediates the fusion of the viral and
target cell membranes [18,19,20]. The high potential energy and
level of exposure of the Envs create opportunities for premature,
irreversible inactivation by small molecules . In addition, entry
inhibitors may also block viral interaction with the host receptors
or interfere with critical conformational transitions of the envelope
proteins during membrane fusion.
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Several inhibitors of the HIV-1 Envs have been developed,
targeting different sites either on the Envs or on the co-receptors
that are required for virus entry. A few small molecules such as
NBD-556 and BMS-378806 (BMS-806) interact with gp120 and
prematurely trigger or interfere with conformational changes in
the Envs, respectively [22,23,24,25,26,27,28,29]. Small-molecule
ligands of CCR5 and CXCR4 bind to the cognate coreceptor
leading, in most cases, to a conformation that is not recognized by
HIV-1 Env, and less frequently, to coreceptor internalization
[30,31,32,33,34,35,36]. The gp41-derived peptide T20 competes
with the gp41 heptad repeat 2 (HR2) region in the intact Envs on
virions and blocks subsequent steps required for membrane fusion
. Several of these inhibitors are very potent and two of them
(T20 and Maraviroc, a CCR5 ligand) have been approved for
treatment of HIV-1 infection. However, under the selective
pressure of these molecules, resistant variants of HIV-1 eventually
emerge, impeding long-term efficacy of the compounds and
underscoring the urgent need for new and broad-range inhibitors
[38,39,40,41,42]. Inhibitors that impose a high genetic barrier to
the development of resistance could lead to novel treatment or
prophylactic options. Such inhibitors could also serve as extremely
useful probes to study the poorly understood transitions of the
HIV-1 Envs to different conformational intermediates along the
virus entry pathway.
The ability of the HIV-1 Envs to mediate membrane fusion at
neutral pH results in Env-expressing cells fusing with receptor-
expressing cells to form multinucleated syncytia [43,44]. Cell-cell
fusion assays are attractive tools for identifying potent inhibitors of
HIV-1 Env function. Such assays mimic the entry steps of HIV-1
into cells, and can measure membrane-fusing activity, as well as its
inhibition, with high sensitivity [43,44,45,46,47,48,49,50,51,52].
The assay also simulates the multiple Env-receptor interactions
that occur during direct transmission of the virus from one cell to
another, and offers a unique system to study and potentially inhibit
this mode of transmission [53,54,55]. Cell-cell fusion systems are
typically composed of two types of cells: effector cells that express
the HIV-1 Envs and target cells that express the CD4 receptor and
CCR5 (or CXCR4) coreceptor. During cocultivation, HIV-1 Envs
expressed on the surface of effector cells bind receptor-bearing
target cells, and fuse the membranes of the cells, leading to the
formation of multinucleated syncytia [43,44]. The fusion event can
be readily detected by inducing expression of a reporter protein in
the target cells. Two different cell-cell fusion assays have been
independently developed for large-scale screening [56,57]. Al-
though they are effective and robust, both can benefit from further
improvement in their design and use. The first system utilized
effector cells that stably express Envs on the surface of Chinese
hamster ovary (CHO) cells; however, because long-term Env
expression may exert subtle cytotoxic effects, the proportion of
correctly folded and processed Envs on the surface of such effector
cells may diminish with time . An intensive screen with this
system has successfully identified the potent inhibitor PF-68742;
however, a single residue change, Gly514Arg (standard HXB2
numbering) in gp41, confers resistance to the compound . The
second assay was configured as two systems, one for screening
CCR5-mediated entry inhibitors and the other for screening
CXCR4-mediated entry inhibitors . Screening a library of
compounds with the CCR5-mediated system and excluding any
compounds that also decreased the CXCR4-mediated fusion
identified CCR5-specific inhibitors. While effective, this strategy
would clearly fail to identify compounds with broad-range activity
that would be expected to inhibit both CCR5 and CXCR4-
mediated entry. Here we present a cell-cell fusion system that
applies a different strategy and carries a built-in capability to
measure non-specific inhibition; the system incorporates the most
advanced elements in a cell-cell fusion system. Each protein is
expressed from a codon-optimized gene, and response elements
are tightly regulated and have low background activity. The
system is robust, has high dynamic range, and can sequentially
measure the inhibition potency and specificity of each compound
in the same cells.
Design of cell-cell fusion system
We have designed and built a unique cell-cell fusion system that
incorporates several features optimized for the study of HIV-1
Env-mediated membrane fusion and its inhibition. Cell-cell fusion
occurs when effector cells expressing HIV-1 Envs are cocultivated
with target cells that express the appropriate receptors. For certain
applications, constitutive expression of HIV-1 Envs may be
important, but Env-related toxicity in cells may counterselect cells
that stably express high levels of correctly processed Envs. To
avoid such an outcome, we have used an inducible Env expression
system that utilizes a tetracycline-controlled transactivator (tTA)
protein and is tightly regulated by doxycycline (Dox, a tetracycline
analogue) [59,60]. Effector cells were generated by transferring the
codon-optimized HIV-1 env gene under the control of a tTA-
responsive promoter into HeLa-TetOff cells, which constitutively
express optimal levels of tTA (Figure 1). The tTA-mediated
transcription of Env can be turned on or off by removing Dox
from or adding Dox to the medium, respectively. This biological
switch ensures tight control of Env expression, which is induced
only immediately prior to the fusion assay, thus avoiding
detrimental effects of Envs during propagation of the effector cells.
To minimize the complexity of the system, the Env-inducing
tTA protein in the effector cells was also used to activate reporter
gene expression in the target cells (Figure 1). For this purpose, the
firefly luciferase (F-luc) gene, under the control of a tTA-
responsive promoter, was transferred into target cells that express
CD4 and CCR5 receptors. Thus, fusion of the effector and target
cells enables the tTA to diffuse from the effector cell to the target
cell and activate the expression of F-Luc protein. F-Luc activity is
proportional to the extent of cell-cell fusion.
Stable effector and target cells
Three different vectors were tested for the ability to express two
different primary HIV-1 Envs transiently in effector cells.
Transfected cells were also tested for membrane-fusing activity
by cocultivation with target cells (Figure S1 and Method S1).
Based on these results, plvx-AD8 and pGL4.2-JRFL, which
express the Envs of two primary HIV-1 isolates, AD8 and JR-FL,
respectively, were selected. These plasmids were used to generate
the stable HeLa-AD8 (H-AD8) and HeLa-JRFL (H-JRFL) cells,
respectively. Expression of Envs was detected in both stable
effector cells, and was controlled by the presence or absent of Dox
in the medium (Figure 2A). Similarly to transient expression
(Figure S1), stable Env expression from the pGL4.2 vector resulted
in higher Env levels relative to expression from plvx-AD8, and
allowed the detection of shed gp120 in the supernatant of the
Two different target cell lines were generated by lentivirus-
mediated gene transfer of the F-Luc gene, under the control of the
tTA-responsive promoter. Both cells express human CD4 and
CCR5. Cf2Luc4#18 cells are canine Cf2Th cells, whereas CEM-
R5Luc1#21 cells are human T lymphocytes. Both were derived
from single clones and were selected for their low background of
fusion with Env-expressor cells in the presence of Dox and high
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signal with Env-expressing cells (in the absence of Dox). Flow
cytometric analysis of CD4 and CCR5 levels on the surface of
these cells showed that Cf2Luc4#18 cells express low-to-moderate
levels of CD4, but high levels of CCR5. In contrast, CEM-
R5Luc1#21 cells express high levels of CD4 and lower levels of
CCR5 (Figure 2B). Thus, different combinations of receptor levels
are represented on the surface of these two cell lines.
To verify the ability of stable effector cells to fuse with stable
target cells, we directly visualized the fusion events using a
fluorescent protein as a reporter for fusion. This method allowed
us to distinguish fusion events from any cell aggregates. For this
purpose, we used target cells that were stably transfected with a
plasmid expressing a green fluorescent protein (GFP) reporter
protein under the control of a tTA-inducible promoter. Stable
GFP-based Cf2Th-CD4/CCR5 target cells (Cf2-tetO-GFP) were
cocultivated in the absence of Dox with either H-AD8 or H-JRFL
effector cells, resulting in recognizable syncytia that were GFP-
positive. H-JRFL cells were more fusogenic than H-AD8, as
indicated by the number and extent of the fusion events. No
syncytia could be detected when Dox was added to the medium.
Notably, the pattern of fluorescence during fusion was quite
different and more disperse than the pattern observed during
transient expression of GFP in the target cells (Figure 3). This may
be attributed to the ability of GFP to diffuse to all cells that form
the syncytium, extending beyond the limits of a single cell.
Effect of assay parameters on the fusion readout
The effects of varying the number of cells, time of incubation
and DMSO concentration on the assay readout were evaluated
with the different effector and target cell types. To study the effect
of changes in the number and ratio of the cells, the number of the
two target and two effector cells was varied in the fusion assay
(Figure 4). Assay readout was robust over a range of target:effector
cell ratios, and generally increased with higher numbers of both
effector and target cells. For Cf2Luc4#18 target cells, the fusion
readout with H-JRFL cells was generally higher and more readily
saturated than that with H-AD8. Cell-cell fusion was also
monitored over time with different combinations of effector and
target cells (Figure 4E). Fusion of Cf2Luc4#18 target cells with H-
JRFL cells showed rapid fusion kinetics and reached a higher
maximum compared to fusion with H-AD8 cells. Fusion of CEM-
R5Luc1#21 target cells with H-AD8 cells exhibited rapid kinetics
and a very low background. In all cases, the assay readout was
completely abolished by Dox, with only a slight increase in
background during the time course.
The effect of DMSO (dimethyl sulfoxide), which is routinely
used to dissolve chemical compounds, on the cell-cell fusion assay
was also studied. For the fusion assay using Cf2Luc4#18 target
cells, concentrations of up to 1% DMSO did not significantly
change the assay readout, demonstrating a high tolerance of the
system to this solvent and its suitability for use in large-scale
screening. Interestingly, the fusion assay using CEM-R5Luc1#21
target cells tolerated DMSO concentrations as high as 2%, and
moderate concentrations of DMSO resulted in higher assay
readouts (Figure 4D).
Levels of control on the cell-cell fusion system
The use of Dox enables two levels of control in the cell-cell
fusion system. tTA activates the expression of HIV-1 Envs in
effector cells, as well as the expression of F-Luc in target cells after
productive cell-cell fusion. Thus, regulation of tTA activity by Dox
enables two types of controlover the fusion process: 1) manipulation
of the level of Env expression in the effector cells by modifying Dox
concentrations during induction; and 2) manipulation of the fusion
readout by modifying Dox concentrations during cell-cell fusion.
The first type of control was tested by inducing H-AD8 cells to
express differentlevels ofEnvs byusing decreasingconcentrations of
Dox; the cells were then washed to remove any Dox and incubated
with Cf2Luc4#18 target cells in the absence of Dox (Figure 5). The
extent of fusion after 7.5 hours correlated with the level of Env
expression; i.e., high fusion readout was associated with low Dox
concentrations. This effect was temporary and substantially
decreased with a longer incubation time of 22.5 hours; such an
outcome was expected because full expression of HIV-1 Envs
occurred beginning at the time of cocultivation of effector cells and
target cells, regardless of the induction condition. The second level
of control involved modification of the fusion readout when Dox
was added during the fusion process. For both types of effector cells,
addition of Dox during the cocultivation of effector and target cells
decreased the fusion readout in a dose-dependent manner, with
almost complete suppression at 1 ng/ml. Accordingly, addition of
2 mg/ml Dox, which completely abolished tTA-related F-Luc
expression, was used in our experimental system to define the
background level of cell-cell fusion.
Dual reporter system
A potential problem in using the cell-cell fusion assay for large-
scale screening is that the final readout could be decreased by
compounds that have non-specific effects on cell viability, or by
compounds that specifically inhibit the tTA-based induction
system. Such unrelated effects can be mistakenly attributed to
Env-specific inhibition, which also decreases the assay readout. To
recognize such false positives, we introduced a tTA-regulated gene
encoding a second reporter, Renilla luciferase (R-Luc), into H-
Figure 1. Assay scheme for HIV-1 Env-mediated cell-cell fusion.
An activator (tTA) is used for inducible expression of the HIV-1 Envs in
the effector cells, allowing Env expression to be turned on by the
removal of Dox from the medium. When effector and target cells are
cocultivated, cell-cell fusion enables the diffusion of tTA to the target
cell. This activates the transcription of the F-Luc reporter gene, which is
also under the control of a tTA responsive promoter. F-Luc activity can
be measured and is quantitatively related to the extent of fusion.
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Figure 2. Expression of HIV-1 Envs in effector cells and HIV-1 receptors in stable target cells. A. The H-AD8 and H-JRFL cells were
induced for 48 hours and then metabolically labeled with35S overnight in the absence of Dox. Control cells were treated similarly but 2 mg/ml Dox
was present in the medium at all times. Cell lysates and supernatants were subjected to radioimmunoprecipitation and analysis on polyacrylamide
gels. B. Levels of expression of CD4 and CCR5 receptors on the two target cells were analyzed by flow cytometry. Cells were stained with secondary
antibody alone as a control, or with either OKT4 (anti-CD4) antibody or 2D7 (anti-CCR5) antibody followed by the secondary antibody. Color codes
are identical for both target cells. MFI, mean fluorescence intensity.
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AD8 effector cells (generating H-AD8#15Ren cells, Figure 6A). In
this setting, induction of the Envs by removal of Dox also induces
the expression of R-Luc in the effector cells. Following effector-
target cell cocultivation, both luciferase activities can be measured
sequentially within the same cell lysate. F-Luc activity measures
the extent of cell-cell fusion, whereas R-Luc activity evaluates off-
target effects. To assess the ability of this system to distinguish
specific fusion inhibitors from unrelated or cytotoxic ones, we
tested different compounds. All four HIV-1 entry inhibitors (T20,
Maraviroc, RPA and NBD-556) exhibited specific inhibition with
low fusion (F-Luc) and high specificity (R-Luc) signals when
compared to a control fusion assay without inhibitors (Figure 6B).
The efficient entry inhibitors T20 and Maraviroc were very potent
and led to almost complete inhibition without any substantial off-
target effects. The weak entry inhibitor NBD-556 was less
effective, and inhibited approximately 50% of the fusion activity
at a concentration of 9.1 mM. Low inhibition by the OKT4 anti-
CD4 antibody was observed with CEM-R5Luc1#21 target cells
but not with Cf2Luc4#18 target cells. Low inhibition of HIV-1
Env-mediated cell-cell fusion has been previously observed for the
highest OKT4 concentrations used in a different assay system
. Importantly, specific inhibition was documented for
inhibitors that are directed against different components of the
HIV-1 entry process, including gp41 (T20), gp120 (NBD-556),
Figure 3. Direct visualization of cell-cell fusion with stably integrated and inducible GFP gene in target cells. The H-AD8 and H-JRFL
effector cells were incubated separately with Cf2-tetO-GFP cells; forty hours later, cells were visualized under a fluorescence microscope. Images were
acquired for target and effector cells alone and after cocultivation. The phase contrast and fluorescence images are shown for each culture, except for
the transiently transfected cells. Cf2Th-CD4/CCR5 cells that were transiently cotransfected with the plvx-tetO-GFP and tTA expression plasmids (plvx-
TetOff Advanced, Clontech) were visualized after 24 hours and are shown in the right top panel.
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CCR5 (Maraviroc) and CD4 (RPA). In contrast, the cytotoxic
translation blocker cycloheximide and Dox, which controls the
expression of both luciferases, decreased both F-Luc and R-Luc
signals. A control antibody and Efavirenz, as expected, did not
have any effect on the activity of either luciferase. Thus, the assay
is capable of distinguishing a specific inhibitor of HIV-1 Env-
mediated membrane fusion from off-target inhibitors.
Statistical analysis showed that the fusion readout as well as the
expression of R-Luc were reproducible in the dual reporter system
(Figure 7). Low variation was observed for readouts from replicate
wells, and incubation with Dox consistently and thoroughly
suppressed both luciferase activities. The calculated Z’-factor was
high for both target cells, demonstrating their suitability for large-
scale screening. A slightly higher value with lower standard
deviation was observed for Cf2Luc4#18 target cells compared
with the values for CEM-R5Luc1#21 cells.
Validation of the system with known inhibitors
The ability of the cell-cell fusion system to monitor inhibition
efficiency as well as specificity can be utilized to measure the
Figure 4. Effects of different assay parameters on the fusion readout. A, B. The influence of changes in the number of Cf2luc4#18 target
cells (designated Cf2 cells), and either H-AD8 (A) or H-JRFL (B) effector cells, on the readout of the cell-cell fusion assay are shown. Color codes are
identical for both panels. C. Same as (A) but with CEM-R5Luc1#21 target cells. D. The effect of different concentration of DMSO on the cell-cell
fusion assay. E. Time course of fusion of H-AD8 and H-JRFL cells with Cf2Luc4#18 target cells and of fusion of H-AD8 cells with CEM-R5Luc1#21 cells.
Results are representative from those obtained in three independent experiments.
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inhibitory and cytotoxic concentrations of a specific inhibitor,
enabling assessment of the therapeutic index. Moreover, such
measurements can be used to evaluate the contribution of any
cytotoxiceffects to compound inhibitionat anygiven concentration.
To evaluate this capability, the dose-response profiles of three
known inhibitors were studied, using both target cells (Figure 8).
The CEM-R5Luc#21 cells were more sensitive to inhibition by all
three inhibitors and the IC50values closely matched published
antiviral data (Figure 8B). Inhibition of cell-cell fusion by Maraviroc
was tightly related to the level of CCR5 expression on the target
cells, as previously published . For fusion with the low-CCR5-
expressing CEM-R5Luc1#21 target cells, the calculated IC50was
about 3 nM, whereas this value increased about 100-fold for the
high-CCR5-expressing Cf2Luc4#18 cells. Less profound differ-
ences between the assay results obtained with different target cells
were observed for inhibition by T20 and BMS-806. Inhibition of
fusion with CEM-R5Luc1#21 target cells was about 3-fold more
efficient for T20 and about 6-fold more efficient for BMS-806,
Luc inhibitory activity of each compound was also evaluated for all
tested concentrations and was used to generate a ‘‘cytotoxic’’ curve
(Figure 8). High concentrations of T20 and Maraviroc appeared to
be slightly toxic in the assay in which HeLa cells and Cf2Luc4#18
cells were cocultivated, as indicated by the small decrease in R-Luc
activity. Except for this effect, none of the compounds was
significantly cytotoxic and no substantial decrease in R-Luc activity
was observed at any tested concentration. These results confirm the
high therapeutic indices of T20, Maraviroc and BMS-806.
We have developed an advanced cell-cell fusion system with a
unique configuration in which the tTA transactivator was
exploited to regulate expression of all components including
HIV-1 Envs, F-Luc and R-Luc. Cocultivation of Env-expressing
effector cells and receptor-expressing target cells resulted in robust
and reproducible cell-cell fusion. The dual system effectively
distinguished specific fusion inhibitors from non-related ones, and
reliably measured inhibitory concentrations as well as cytotoxicity
for known fusion inhibitors.
The tetracycline–inducible expression system was selected
because it is well characterized and offers very tight regulation,
with low background levels of transcription and high expression
levels . Interestingly, Env expression from the non-lentiviral
vector pGL4.2-JRFL was consistently higher than Env expression
from the lentiviral vector plvx-JRFL in both transiently and stably
transfected cells. Although the tTA promoter and Env sequences
are identical in both vectors, the flanking sequences in the vector
may suppress or enhance tTA-mediated transcription; moreover,
distinct integration sites in the stable cell lines may exert different
effects on transcription.
The specific design of our cell-cell fusion system offers several
advantages: 1) the use of inducible stable cell lines avoids Env-
related toxicity and provides consistent expression of HIV-1 Envs
relative to repeated transient transfections, which, in addition to
being more variable, are time- and resource-consuming; 2) HIV-1
Env expression is tightly regulated and derived from a codon-
optimized gene, alleviating the requirement for the HIV-1 Rev
protein in the system; 3) tight control of tTA allows a complete
shutoff of transcription of all components of the system, as well as
the ability to control Env expression level prior to the cocultivation
of effector and target cells; 4) R-Luc activity can be used as a
surrogate for the level of Env expression on the effector cells
because both Envs and R-Luc are regulated by the same tTA
protein in these cells; 5) The system can distinguish tTA inhibitors,
which will result in off-target effects, from specific fusion inhibitors
because R-Luc activity is regulated by tTA; and 6) dual
measurements of luciferase activity allow the evaluation of both
efficiency and specificity of inhibition in the same cells under the
identical experimental conditions, avoiding potential errors
associated with measuring these two properties separately. The
only limitation of our system is the inability to distinguish fusion
inhibitors from specific F-Luc inhibitors. F-Luc inhibitors will
lower F-Luc activity without a significant decrease in R-Luc
activity, and therefore will be scored as specific fusion inhibitors
(false positives). Nevertheless, such compounds are presumably
very rare, are easily identifiable in secondary assays, and thus
should not significantly compromise the utility of the system to
Some of the above features of our system have been previously
used in transient or stable fusion assays. However, these systems
have not incorporated all of these features into a single complete
system; moreover, the manner in which these elements were
utilized differs from ours. One assay monitored the fusion of
Figure 5. Two levels of control on the fusion assay. A. The levels of expression of HIV-1 Envs in the H-AD8 effector cells were controlled by
varying the concentration of Dox during induction. Cells were washed prior to fusion, and allowed to fuse with Cf2Luc4#18 cells in Dox-free medium,
initiating full induction of HIV-1 Envs. The fusion readout is shown after 7.5 and 21.5 hours. B. The level of expression of HIV-1 Envs in H-AD8 and H-
JRFL cells, as well as that of F-Luc in the Cf2Luc4#18 target cells, was suppressed during fusion by adding Dox into the medium. The results shown
are representative of those obtained three independent experiments.
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Figure 6. Design and testing of a dual reporter assay measuring inhibition efficiency and specificity. A. A dual reporter system was
designed by incorporating the R-Luc gene, under the control of a tTA-responsive promoter, into effector cells. When these cells are fused to target
cells, two readouts are measured sequentially: F-Luc activity measures the extent of cell-cell fusion and R-Luc activity measures any off-target effect.
Specific fusion inhibitors are expected to give low F-Luc and high R-Luc activities, whereas unrelated inhibition results in low activities of both
luciferases. B. Various compounds were tested with the dual reporter system using H-AD8#15Ren effector cells and CEM-R5L1#21 target cells. Both
F-Luc and R-Luc activities were measured and normalized to those activities seen in cells without any inhibitor. The final concentration of DMSO
during the cell-cell fusion assay was 0.1% unless otherwise indicated. T20, T20 peptide (118 nM); Mar, Maraviroc (1.18 mM); CHX, cycloheximide
(100 mg/ml); RPA, RPA-T4 (anti-CD4 antibody, 1 nM); OKT, OKT4 (anti-CD4 antibody, 1 nM); Con.Ab, Control antibody (1 nM); NBD, NBD-556 (9.1 mM);
Dox, doxycycline (2 mg/ml); EF, efavirenz (an HIV-1 reverse transcriptase inhibitor, 312 nM).
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transiently transfected 293T cells ; in this case, both
transfection efficiency and cytotoxicity were measured with a
single R-Luc marker, and thus the contribution of each to the total
effect was unknown. A tTA-based system has been used for
transient expression of Envs and for detection of fusion inhibition
. Another fusion system with inducible expression of HIV-1
Envs has also been reported . In this system, Dox was used for
induction and was required throughout the fusion assay.
Coexpression of HIV-1 Rev was required for Env expression,
and HIV-1 Tat was used as a transactivator to trigger F-Luc
expression in the target cells. Identifying fusion inhibitors in this
setting requires an additional step to confirm that the compounds
do not inhibit either HIV-1 Rev or Tat. In contrast to our system,
this earlier assay did not contain a built-in ability to measure off-
Our cell-cell fusion system, like other fusion systems, is focused
on a single step in the HIV-1 life cycle. Such a strategy will not
identify inhibitors of other steps in the virus life cycle, but has
several advantages. Compared with more comprehensive antiviral
assays, the more focused cell-cell fusion assay requires a shorter
time of incubation, may be less sensitive to non-specific effects, and
allows the target of any inhibitor to be easily identified. In
addition, the cell-cell fusion system does not require the use of
special biosafety containment facilities, as no infectious virus is
Our dual reporter system was successfully tested against a wide
range of inhibitors and control molecules. The system detected
known fusion inhibitors and underscored their specificity, whereas
control agents did not significantly affect cell-cell fusion. Specific
inhibition was documented for inhibitors that target different Env
or receptor components of the HIV-1 entry process; for different
types of inhibitory molecules such as chemical compounds,
peptides and antibodies; and for inhibitors with diverse levels of
efficiency [ranging from an IC50of ,10 mM (NBD-556) to ,3nM
(Maraviroc)]. Non-related effects of cycloheximide and tTA-
inhibitors were clearly categorized as off-target, as they completely
abolished the activity of both luciferases. Measurements of
inhibitory concentrations and cytotoxicity of known inhibitors
validated the utility of the fusion system. The system was efficiently
inhibited in a dose-response manner by all known inhibitors, with
different efficacy for the two target cells types tested. CCR5-
directed Maraviroc was the most potent inhibitor of fusion to
CEM-R5Luc1#21 cells and a less efficient inhibitor for fusion to
Cf2Luc4#18 cells. Such differences in potency are expected, since
the lower number of target CCR5 molecules on CEM cells,
relative to those on Cf2Th cells, enables effective inhibition by
even low concentrations of Maraviroc. Our results suggest that the
distinct properties of the two target cells available for this cell-cell
fusion system may exhibit different levels of sensitivity for the
detection of particular types of inhibitors. CEM-R5Luc1#21 cells
grow in suspension and are derived from human T-cells that are
closely related to authentic target cells during HIV-1 infection;
fusion with CEM-R5Luc1#21 cells is inhibited very efficiently by
known fusion inhibitors. Cf2Luc4#18 are adherent cells, and are
less sensitive to fusion inhibitors, but exhibited a slightly higher Z’-
factor with less variation.
Adaptation of the dual reporter system to high-throughput
screening in a 384-well format may require additional optimiza-
Figure 7. Statistical analysis of the dual reporter system with two different target cells. A. Distribution of F-Luc and R-Luc signals
obtained in the cell-cell fusion assay conducted with or without 2 mg/ml Dox. Readouts of fusion without Dox are from 35 wells of a single plate and
those with Dox are from 35 wells from multiple plates. B. Statistical analysis for the precision of the system. Data from 5 (CEM-R5Luc1 cells) or 4
(Cf2Luc4#18) independent experiments were used to calculate the Z’-factor of the assay for each luciferase. Means 6 standard deviations are shown
for each cell type.
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PLoS ONE | www.plosone.org9 November 2011 | Volume 6 | Issue 11 | e26731
Figure 8. Dose-response curves for inhibition efficiency and specificity of known HIV-1 entry inhibitors. A. The gp41-directed T20
peptide, gp120-directed BMS-806, and CCR5-directed Maraviroc were tested for inhibition and off-target effects with the dual reporter system using
two different target cells, CEM-R5Luc1#21 (left) and Cf2Luc4#18 (right). Inhibition data were fitted to the logistic equation and the IC50value for
each tested compound and each target cell type was calculated. R2.0.96 for all curves. Red lines display relative R-luc activity and indicate the
specificity of a compound; green lines are fitted curves for relative F-luc activity and indicate the inhibition of cell-cell fusion for each compound. The
results shown are representative of those obtained in three or four independent experiments, each performed with more than three replicates. B.
Reported IC50values for the three inhibitors in antiviral assays [24,27,37,62,71,72] (inhibition of the HIV-1MNisolate by BMS-806 was reported to be
743 nM  and this atypical value is excluded from the table).
Screening Assay for HIV-1 Entry Inhibitors
PLoS ONE | www.plosone.org 10November 2011 | Volume 6 | Issue 11 | e26731
tion of cell numbers to adjust for the lower volumes. In addition,
commercial luciferase substrate solutions, such as Steady-Glo
(Promega), which are designed to have a long half-life for batch
processing, have lower luminescence intensities in comparison to
other formulations that are designed for high sensitivities (Steady-
Glo Technical Manual, Promega). Using such a substrate resulted
in lower readouts than the luciferase substrate that was used in our
study (data not shown). Moreover, because some decrease of signal
readout was observed after long-term propagation of the effector
cells, the use of low passage cells is recommended. Preliminary
data from scaled-up pilot studies indicate that the cell-cell fusion
assay is robust and underscore the importance of using a control
system for excluding off-target compounds.
In summary, our assay is simple and efficient. The system has
high dynamic range, tolerates DMSO, is precise (high Z’-factor
scores) and has been validated with a panel of reference inhibitors
directed against different steps in the fusion process, yielding
inhibitory concentrations consistent with published data. The dual
reporter system can be used for detailed characterization of
inhibition versus cytotoxic properties of entry inhibitors. In
addition, the system is suitable for a high-throughput screen of
chemical compounds and adds a unique layer to the already
existing cell-cell fusion systems. We are currently developing
additional cell-cell fusion assays that utilize Env derived from
different primary HIV-1 isolates. Combining the screening with
secondary assays that utilize cells expressing Env from different
HIV-1 isolates may identify broad-range fusion inhibitors that can
generate novel lead compounds for further development as
therapeutics or prophylactic microbicides.
Materials and Methods
Reagents and antibodies
Maraviroc and T20 were obtained from the AIDS Research
and Reference Reagent Program, Division of AIDS, NIAID, NIH.
Cycloheximide was from Sigma. RPA-T4, 2D7 and control
isotype antibodies were purchased from BD Biosciences; OKT4
was obtained from eBiosciences.
HeLa-TetOff cells stably expressing the tetracycline-controlled
transactivator were purchased from Clontech, and maintained in
DMEM supplemented with 10% heat-inactivated fetal calf serum
(FCS), 2mM L-glutamine, 100 units/ml penicillin, 100 mg/ml
streptomycin, and 100 mg/ml geneticin (all from Gibco). The
canine Cf2Th-CD4/CCR5 cell line stably expressing the human
CD4 receptor and CCR5 coreceptor  was maintained in the
same medium supplemented with 400 mg/ml geneticin and
200 mg/ml hygromycin (Gibco). Human embryonic kidney
293T/17 cells (designated 293T) were obtained from the
American Type Culture Collection (ATCC) and maintained in
CEM.NKR-CCR5 cells were obtained through the AIDS
Research and Reference Reagent Program, Division of AIDS,
NIAID, NIH from Dr. Alexandra Trkola [64,65,66], and were
maintained in RPMI-1640 supplemented with 10% FCS, 2mM L-
glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin.
The plvx-AD8 plasmid was generated by PCR amplification of
the HIV-1AD8env gene from pcc-AD8on (a gift from Dr Xinzhen
Yang, Beth Israel Deaconess Medical Center) and cloning the
PCR product into the BamHI/EcoRI restriction sites of the plvx-
Tight-Puro vector (Clontech). The HIV-1AD8 env sequences
encoding the first 610 residues of Env are codon-optimized and
enable Env expression without the need for HIV-1 Rev. The plvx-
JRFL plasmid was generated in a similar manner, by cloning the
fully codon-optimized version of HIV-1JRFLenv into plvx-Tight-
Puro. The pGL4.22-JRFL plasmid was generated by digesting
plvx-JRFL with XhoI/EcoRI and subcloning the ,2900 bp
fragment (containing the tetracycline-controlled promoter and
pGL4.22[luc2CP_Puro] (Promega). The pGL4.78_tetO_Renilla
plasmid was generated by PCR amplification of the tetracycline-
controlled promoter and cloning it into NheI/HindIII restriction
sites in pGL4.78[hRlucCP_Hygro] (Promega). The plvx-tetO-
GFP plasmid was generated by cloning the GFP gene into plvx-
Tight-Puro-Luc (Promega), replacing the luciferase gene with the
GFP gene. All plasmids were sequenced to verify the correct
sequence of the inserts.
Generation of stable cell lines
Stable gene transfer of the AD8 env gene was mediated by
pseudoviruses that were prepared by transfecting 293T cells with
plvx-AD8, and a mix of helper virus expression plasmids,
according to the manufacturer’s instructions (Clontech). Pseudo-
viruses were used to infect HeLa-TetOff cells that were then
selected with 1 mg/ml puromycin to generate H-AD8 cells. H-
JRFL cells were generated by transfecting the HeLa-TetOff cells
with pGL4.2-JRFL, followed by selection with 1 mg/ml puromy-
cin. From the first day after infection (or transfection), 2 mg/ml
Dox (Clontech) was added to the growth medium to avoid any
detrimental effect caused by the expression of the HIV-1 Envs.
More than 20 different single clones were isolated after
propagating H-AD8 cells in 96-well plates under limiting dilution
conditions, and each clone was tested for its fusion activity. H-
AD8#15Ren was generated by transfecting AD8 clone #15,
which demonstrated the most efficient fusion to target cells in pilot
assays, with the pGL4.78_tetO_Renilla plasmid and subsequent
selection with 200 mg/ml hygromycin.
Cf2luc4 and Cf2-tetO-GFP target cells were prepared in a
manner similar to that described above, using either plvx-Tight-
Puro-Luc plasmid (for Cf2luc4 cells, Clontech) or plvx-tetO-GFP
(for Cf2-tetO-GFP cells) to prepare pseudoviruses. Viruses were
used to infect Cf2Th-CD4/CCR5 cells that were subsequently
selected with 4 mg/ml puromycin. CEM-R5Luc1 target cells were
generated by infecting CEM.NKR-CCR5 cells with pseudoviruses
that were prepared with the plvx-Tight-Puro-Luc plasmid and
subsequent selection with 1 mg/ml puromycin. Single clones were
isolated as described above for H-AD8 clones. Cf2Luc4#18 and
CEM-R5Luc1#21 exhibited high fusion activity and the lowest
background in the cell-cell fusion assay.
Half a million cells were analyzed by flow cytometry as
previously described , but with primary antibody incubation
for 30 min, and secondary antibody (R-Phycoerythrin-conjugated
goat anti-mouse IgG, Invitrogen) incubation for 10 min, both at
room temperature. Cells were analyzed with a BD FACSCanto II
flow cytometer (BD Biosciences) and results were displayed using
Flow Jo software (Tree Star).
Cell-cell fusion assay
Effector cells were washed 3 times with PBS, trypsinized, and
16106cells were seeded at a concentration of 16105/ml in
DMEM medium without phenol red, containing Tetracycline-
approved FBS (Clontech) and supplemented with 100 mg/ml
geneticin and 1 mg/ml puromycin (200 mg/ml hygromycin was
Screening Assay for HIV-1 Entry Inhibitors
PLoS ONE | www.plosone.org11 November 2011 | Volume 6 | Issue 11 | e26731
also added for H-AD8#15Ren cells). After incubating for 3 hours
at 37uC, the medium was replaced by fresh medium to remove any
traces of Dox and to enable full induction of HIV-1 Env
expression. Cf2Luc4#18 target cells were seeded at 16105/ml
in the same medium as above, but supplemented with 400 mg/ml
geneticin, 4 mg/ml puromycin and 200 mg/ml hygromycin. After
overnight incubation (,24 hours), effector and target cells were
detached with 5 mM EDTA in PBS or with a minimal amount of
trypsin, washed once with PBS and then added sequentially to a
96-well B&W isoplate (PerkinElmer) in the above medium without
any selection antibiotics. After an overnight incubation at 37uC,
the medium was aspirated, 30 ml of passive lysis buffer (Promega)
was added to each well, and F-Luc activity was measured with a
Centro LB 960 luminometer (Berthold Technologies, TN, USA).
One hundred microliters of assay buffer (15 mM MgSO4, 15 mM
KH2PO4/K2HPO4pH 7.8, 1 mM ATP and 1 mM DTT) was
injected, followed by a 50 ml injection of 1 mM luciferine (BD
Biosciences); luminescence was measured for 10 sec. For dual
assay measurements, this procedure was followed by injecting
50 ml of quench-substrate solution [2.2 M NaCl, 4.4 mM EDTA
pH 8, 0.44 M KH2PO4/K2HPO4 pH 5.1, 0.88 mg/ml BSA,
2.6 mM sodium azide and 2.9 mM coelenterazine (Promega)] and
measuring R-Luc activity for 10 sec . When CEM-R5luc1#21
target cells were used for fusion, they were spun down and
resuspended in RPMI-1640 medium containing Tetracycline-
approved FBS (Clontech) without Phenol Red prior to the assay.
Approximately 16104target cells were added to each well
containing the same number of pre-incubated effector cells, and
the fusion assay was carried out in either DMEM/RPMI-1640
(50%/50%) or complete RPMI-1640 medium. For the kinetic
experiments, cells were detached with 5 mM EDTA in PBS, lysed
after the specified time of target-effector cell cocultivation, and
frozen at 280uC until luciferase activity was measured. For
inhibition experiments, cells and inhibitors were sequentially
added to the wells, and the cell-cell fusion assay was carried out for
13-15 hours at 37uC. Inhibition data were fitted to the logistic
equation using the nonlinear curve fit module in Origin 8.1
software, as previously described . For statistical analysis, the
Z’-factor was calculated using the fusion readout as the high signal
and fusion in the presence of Dox as the low signal, as described
: Z’ = 1 2 [(3*SDfusion + 3*SDfusion+Dox)/(Meanfusion 2
Meanfusion+Dox)], where SD represents the corresponding standard
deviation of fusion with or without Dox.
Approximately 2–3 6105effector cells (H-JRFL or clone#7 of
H-AD8) were induced for 48 hours at 37uC, metabolically labeled
overnight with 30 mCi
(150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycho-
late, 0.1% SDS, 50 mM Tris, pH 8.0) that contained protease
inhibitor cocktail (Roche). The lysate was cleared and subjected to
immunoprecipitation with serum from HIV-1-infected individuals
followed by SDS-PAGE and autoradiography.
35S and then lysed with RIPA buffer
Effector cells were incubated with Cf2-tetO-GFP target cells, as
described in the fusion assay section above, in 96-well Optilux
black plates with clear bottoms (BD Biosciences). After an
approximately 40-hour incubation at 37uC, cells were visualized
under a Nikon Eclipse Ti-S fluorescence microscope equipped
with a DS-Qi1 camera, and images of live cells were acquired.
ity of HIV-1 Envs expressed by different vectors. A.
Expression of HIV-1 Envs using three different vectors was
measured in HeLa-TetOff cells. Two lentivirus vectors (plvx-AD8
and plvx-JRFL) were used for induced expression of either HIV-
1AD8 or HIV-1JR-FL Envs, respectively, and one nonlentiviral
vector (pGL4.2-JRFL) for induced expression of HIV-1JR-FLEnvs.
HeLa-TetOff Cells were transiently transfected in the presence of
varying concentrations of Dox or without any Dox; cells were
lysed and analyzed by Western blotting (as described in Method
S1). Lane 1, untransfected cells; lane 2, cells transfected with a
control vector (plvx-Tight-Puro); lanes 3-7, cells transfected with
0.8 mg of the indicated plasmid in the presence of specified
concentrations of Dox; lane 8, recombinant HIV-1YU2 gp120
(positive control). B. Effector cells were transiently cotransfected
with the specified plasmid and an R-Luc-based vector for
normalization (as described in Method S1). Cf2Th-CD4/CCR5
cells were transfected with plvx-Tight-Puro-Luc. The cells were
allowed to fuse and F-Luc activity was measured. Fusion was
normalized for transfection efficiency by the R-Luc activity.
Transient expression levels and fusion activ-
We thank Ms. Yvette McLaughlin and Ms. Elizabeth Carpelan for
manuscript preparation. We thank the AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH for providing T20,
Maraviroc, and CEM cells that were used in the study. We thank Dr. S.
Jhaveri, for critically reading the manuscript and for helpful comments; Dr.
B. Pacheco, for the pcDNA-GFP plasmid; Dr. Y. Mao, for the pcDNA-
JRFL plasmid; Dr. X. Yang, for the pcc-AD8on plasmid; and Dr. N.
Madani, Dr. H. Haim, and A.M. Princiotto for helpful discussion and
Conceived and designed the experiments: AH ABS JGS. Performed the
experiments: AH AF DJ JRC AS. Analyzed the data: AH AF ABS JGS.
Contributed reagents/materials/analysis tools: AH AF DJ JRC AS. Wrote
the paper: AH ABS JGS.
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